Cross-linked hyaluronic acids and medical uses thereof

ABSTRACT

Cross-linked hyaluronic acids produced by the reaction of the carboxylic acid groups of hyaluronic acid and a polyamine and the sulfated and hemisuccinylated derivates thereof. The cross-linked hyaluronic acids are useful for various pharmaceutical and medical purposes.

FIELD OF THE INVENTION

The present invention concerns cross-linked hyaluronic acids, optionallyhemisuccinylated or sulphated, the salts thereof with biologicallysuitable or pharmacologically active cations and the complexes thereofwith heavy metals such as copper, zinc and iron.

The invention also concerns the use of said cross-linked hyaluronicacids, salts and complexes in the medical, pharmaceutical and cosmeticfields.

BACKGROUND OF THE INVENTION

Hyaluronic acid is a glycosaminoglycan consisting of disaccharide unitsof D-glucuronic acid andN-acetylglucosamino-2-acetamido-2-deoxy-D-glucose, connected by β (1→3)glycoside bonds.

Natural hyaluronic acid has linear, not cross-linked structure ofmolecular weight ranging from 50,000 to 8,000,000 D or more, dependingon the source and extraction method.

Hyaluronic acid is present in the synovial liquid, connective tissue andvitreous humor of higher animals, as well as in some bacteria.

Compositions of sodium hyaluronate having various molecular weights (inthe form of solutions having different viscosities, gels with differentviscoelastic characteristics, sponges, films or membranes) are used inhuman medicine and surgery for instance as substitutes of synovialliquid, tissular antiadhesive agents, substitutes of vitreous humor,artificial tears, agents for the in vivo tissular re-constitution (forinstance as extra-cellular matrices for the formation of bone segments,following the colonisation of osteoblasts and subsequent calcification;of connective-dermal tissues, following the colonisation offibroblasts), materials for the preparation of artificial skin useful inthe treatment of burns or in plastic surgery; coating agents forbiocompatible vascular prosthesis, carriers of pharmacologically byactive ingredients in controlled-release formulations, etc.

In dermatology and cosmetology, in view of the viscoelastic andmoisturising properties and of the high biocompatibility, saidcompositions are used both as bases for moisturising topicalformulations and as invasive medical-surgical devices (“fillingagents”).

The use of natural, linear hyaluronic acid for said uses is howeverlimited by its in vivo fast degradation by enzymatic systems such ashyaluronidase, glucosidase and glucuronidase, with subsequent decreasein the molecular weight and progressive impairment of the viscoelasticproperties and, generally, of the physical characteristics of the finalcompositions and devices (mechanical strength, elasticity, pore size,)etc.

In order to overcome this problem, mainly with the purpose of increasingthe range of compositions and their applicative flexibility, chemicallymodified hyaluronic acids have been proposed.

Cross-linking with polyfunctional epoxides (U.S. Pat. Nos. 4,716,224,4,772,419, 4,716,154), polyalcohols (U.S. Pat. No. 4,957,744),divinylsulphone (U.S. Pat. Nos. 4,582,865, 4,605,601, 4,636,524),aldehydes (U.S. Pat. Nos. 4,713,448, 5,128,326, 4,582,568),biscarbodiimides (U.S. Pat. No. 5,356,883), polycarboxylic acids(EP-A-718312) has been disclosed.

Said cross-linked hyaluronic acids are used as biomaterials forimplants, prosthesis and medical devices, as controlled-release matricesfor medicaments, as healing, anti-adhesive and dressing agents.

The sulphation of non cross-linked hyaluronic acid is generallydisclosed in U.S. Pat. No. 5,013,724, mainly concerning the sulphationof heparines, heparans and dermatans for use as antithrombotic andanti-coagulant agents.

The hemisuccinylation recreation of hyaluronic acid (HY) has never beendisclosed. An example of this functionalization is disclosed inEP-B-200574, claiming composite biomaterials consisting of succinylatedcollagen and chitosan.

The cross-linking of carboxyalkyl cellulose by means of di- orpolyamines is disclosed in EP-A-566118 (Kimberly Clark Corp) for thepreparation of absorbing materials with HY as cross-linking agent, byheating. Such a method appears to be economically advantageous andsuitable for the large-scale productions required for this kind ofproducts.

EP-A-462 426 (Fidia) discloses perforated biocompatible membranes andtheir uses as artificial skin. Collagen cross-linked with diamines andhyaluronic acid are generically cited as possible materials for saidmembranes.

SUMMARY OF THE INVENTION

It has now been found that new cross-linked hyaluronic acids obtainableby reaction of suitably activated carboxy groups of HY with a polyamine,as well as the salts and complexes with suitable organic or inorganiccations, have advantageous chemico-physical and biological propertiesfor the biomedical and cosmetic uses.

The main chemico-physical and biochemical characteristics of thecompounds of the invention are:

high biocompatibility;

high resistance to enzymatic degradation mainly after sulphation;

high capacity to adsorb water, with formation of visco-elasticcharacteristics dependent on the cross-linking degree as well as onsulphation and/or hemi-succinylation degree;

ability to chelate metal ions such as zinc or copper; said derivativeshaving very good stability.

The biological behaviour is new and surprising; it is known thatsulphation (or supersulphation) of glycosaminoglycans such as heparin,dermatan sulphate, chondroitin and native hyaluronic acid is known toincrease their anti-coagulant properties (inhibition of Xa and IIafactors and/or change of their ratio) with respect to the startingproduct (U.S. Pat. No. 5,013,724).

The compounds of the invention, when sulphated, have a slightanticoagulant activity, whereas it is completely surprising the lack ofplatelet activation and aggregation (measured as antiadhesive activity;P.R.P. model in rabbits subjected to behavioural stress, described in“Abstract IL 15”—International Conference on Advances in Biomaterialsand Tissue Engineering, 14-19 Juin 1998, Capri Italy) both for thecross-linked hyaluronic acid of the invention (with differentcross-linking degrees) and for the corresponding sulphate esters; thisproperty is totally absent in the natural hyaluronic acid and estherderivatives.

No polymeric materials for medical use up to now known apparently sharesthe same property.

DETAILED DISCLOSURE OF THE INVENTION

The invention concerns new cross-linked hyaluronic acids obtainable byreaction of activated carboxylic groups of native linear hyaluronicacid, of extractive or biosynthetic route, with a polyamine,particularly a linear alkyl diamine.

According to a preferred embodiment, the cross-linked hyaluronic acid ofthe invention is further subjected to sulphation and hemi-succinylationprocesses. The obtained products and their salts or complexes haveentirely new properties (for instance, swelling, water motility withinthe gel; chemotactic activity on endothelial cells, viscoelasticproperties).

Said esterification processes are carried out by known methods (use ofreagents pyridine/SO₃; chlorosulphonic acid; succinic anhydride, inhomogeneous or heterogeneous phase, at pH from 6.5 to 8).

Examples of the hemisuccinylation process for collagen are reported inWO 88/10123 and in U.S. Pat. No. 4,493,829.

The polyamine to be used as cross-linking agent according to theinvention is preferably a diamine of formula R₁NH-A-NHR₂ wherein A is aC₂-C₁₀ linear or branched alkylene chain, preferably a C₂-C₆ chain,optional substituted by hydroxy, carboxy, halogen, alkoxy and aminogroups; a polyoxyalkylene chain [(CH₂)_(n)—O—(CH₂)_(n)]_(m) wherein n is2 or 3, m is an integer from 2 to 10; a C₅-C₇ cycloalkyl group; an arylor hetaryl group, preferably 1, 4 or 1, 3 disubstituted benzene; R₁ andR₂, which are the same or different, are hydrogen, C₁-C₆ alkyl, phenylor benzyl groups.

Preferred meanings of A are C₂-C₆ alkylene or a chain [(CH₂),—O—(CH₂)_(n)]_(m). R₁ and R₂ are preferably hydrogen.

The polyamine is reacted with hyaluronic acid or salts thereof, thecarboxylic groups of which have been previously activated.

The activation may be carried out with conventional methods; forinstance, and preferably, those commonly used, in anhydrous aproticsolvent, to form amide bonds in peptide synthesis such ascarbonyldiimidazole; carbonyl-triazole; hydroxybenzotriazole;N-hydroxysuccinimide; p-nitrophenol+p-nitrophenyltrifluoro acetate,chloromethylpyridylium iodide; preferably chloromethypyridylium iodideand like; these activators allow the best yields and the highestreproducibility in terms of cross-linking degree.

The hyaluronic acid is preferably salified with a lipophilic cation, forinstance tetralkylammonium or other lipophilic organic bases able toinduce the suitable solubility in the polar aprotic solvent such asdimethylformamide, tetrahydrofuran or the like.

The transformation of inorganic salts such as sodium into suitableorganic cations may be carried out by well known ion-exchange methods inhomogeneous phase or by precipitation of the acid component, itsrecovery and subsequent salification with the desired organic base.

The activation reaction of the carboxy groups is usually carried out inhomogeneous phase and in anhydrous polar aprotic solvent.

The cross-linking polyamine is added to the solution of the activatedester in the same anhydrous solvent, keeping the temperature from 0 to30° C. The reaction times range from 1 to 12 hours, depending on thepresence of suitable bases such as triethylamine.

In general, the desired final product is recovered by addition of adifferent solvent under reduced pressure, followed by conventionalwork-up.

The cross-linking degree may be comprised within wide limits and may beadjusted by changing the amount of the carboxy-activating agent, theactivation and the cross-linking reactions being practicallyquantitative.

As a consequence, the desired cross-linking degree (C.L.D.: percent ofcarboxylic groups involved in the cross-linking) is perfectlyreproducible, as shown by the N.M.R. data. The final products obtainedunder similar operative conditions have therefore constantcharacteristics.

The starting hyaluronic acid may be any hyaluronic acid having molecularweight from about 5,000 to 8,000,000 D, preferably from 10,000 to200,000 D, extracted from conventional sources or obtainable byfermentation of microorganisms of the group Streptococcus or otherengineered strains.

The cross-linked hyaluronic acid of the invention may be subjected tosulphation reaction with a suitable reagent, preferably thepyridine/sulphur trioxide complex in dimethylformamide.

The reaction is carried out in heterogeneous phase at a temperature of0-10° C. for reaction times ranging from about 0,5 to about 6 hours.

The obtainable sulphation degree may be comprised within wide limits andmay be adjusted by changing the reaction time and the temperature.

Generally, the sulphation degree (defined as eq. Sulphate groups/g) mayrange from 1×10⁻⁶ to 6×10⁻⁶, preferably about 2×10⁻⁶ eq./g for a C.L.D.=0.5.

The cross-linked hyaluronic acid of the invention may also be subjectedto hemisuccinylation reactions in known conditions (aqueousheterogeneous phase, under strong stirring, addition of solid succinicanhydride in subsequent portions, in ratios from 1:1 to 1:5 by weight;keeping the pH from 7 to 8.5 with alkali, at temperatures ranging from 5to 30° C.). The hemisuccinylation degree may be comprised within widelimits depending on the following parameters: reaction time andtemperature; stirring speed of the polyphasic system and addition rateof solid succinic anhydride. By keeping said parameters constant, thereaction gives reproducible products. The cross-linked hyaluronic acids,optionally sulphated or hemisuccinylated, of the invention show theability to form complexes with metal ions such as copper, zinc, iron.

These complexes may be easily obtained by dissolving or by dispersinguntil complete swelling the hyaluronic acid derivative in water andadding under stirring preferably at room temperature, a concentratedsolution of an organic or inorganic salt of copper, zinc or iron, forinstance CuCl₂, ZnCl₂, or Fe₂(SO₄)₃; after 12-24 hours under stirring,the complex is recovered by centrifugation or precipitation followingchange of solvent (e.g. addition of ethanol or acetone) or evaporationunder reduced pressure; the recovered crude product is thoroughly washedwith distilled water so as to remove the excess ion.

The complexes are then freeze-dried.

The content of metal ions depends on the used operative conditions:polymer to ion molar ratios, concentration and pH of the solution;reaction times and particularly the cross-linking degree. It may reachthe maximum volume of 1 metal ion per disaccharide unit not involved inthe cross-linking.

An important advantage of the invention consists in the possibility ofobtaining, by suitably changing the cross-linking degree and/or thesulphation or succinylation degree, hyaluronic acid derivatives in awide range of different forms, characterised by different properties(such as visco-elasticity, metal ions, ability to form hydrogels, films,sponges, mechanical strength etc.).

This allows the use of the hyaluronic acid derivatives of the inventionin several medical and pharmaceutical fields, in the human or veterinaryfield:

1) as intraarticular substitutes of the synovial liquid for thetreatment of osteoarthritic conditions;

2) as vitreous humor substitutes for the treatment of pathologies andside-effects connected to ophthalmic surgery;

3) as base of artificial tears formulation, suited for the therapy ofdry eye;

4) as controlled—release matrices of medicaments (e.g.antiinflammatories, antibiotics, β-adrenergic agonists and antagonists,aldose reductase inhibitors, anti-acne, antiallergic, anti-alopecia,antineoplastic, antiglaucoma, anti-itching, anti-psoriasis,anti-seborrhea, anti-ulcer, antiviral agents, growth factors etc.) bysimple inclusion into the hydrogels obtained from the compounds of theinvention. Alternatively to the in inclusion process, the medicament maybe bound by covalent bonds to the hyaluronic acid matrices, by means of:

a) esterification or amidation of COOH not involved in the cross-linkingwith polyamines, when the medicament is an alcohol or an amine;

b) esterification with the free hydroxy groups of hyaluronic acidderivatives when the medicament has free carboxy groups.

The products under a) may be obtained using the same activation methodof the carboxy groups described above in a carefully anhydrous medium orby transesterification.

5) For the preparation of device for wound or skin ulcers healing inform of films of different thickness, more or less permeable to gases,sponges etc. Said devices preferably contain suitable drugs such asantibiotics, healing factors. They are also useful in the culture ofepithelial cells, keratinocytes etc.;

6) For all the applications for which the use of known hyaluronic acidshas already been proposed, for instance the preparation of solid orsemi-solid forms or moldable form for the production of vascularprosthesis (antiadhesive dressings of blood vessels, artificial heartvalves etc.); of biohybrid organs (artificial pancreas, liver); ofophthalmic products (lens substitutes, contact lens); of otologicalproducts; generally of anti-adhesive implants, to be used in abdominal,gynaecological, plastic, orthopaedic, neurological, ophthalmological,thoracic, otorhinolaryngological surgery; of medical device such asstents, catheters, cannulas and the like.

The uses of cross-linked hyaluronic acid and of biomaterials obtainedtherefrom are known and described, for instance, in WO 97/39788, WO97/22629, WO 97/18244, WO 97/7833, EP 763754, EP 718312, WO 96/40005, WO96/33751, U.S. Pat. No. 5,532,221, WO 95/1165 e EP 320164.

The use of the cross-linked hyaluronic acids of the invention incosmetic dermatology is of particular interest, for instance asmoisturizing agents, bases of various cosmetological formulations,injectable filling agents etc.

The formal products obtained from the cross-linked hyaluronic acidderivatives of the invention may by subjected to sterilisation processes(for instance by heating to 120° C. or by means of ethylene oxide)without any change in the technological properties, which is of course afurther advantage provided by the present invention.

The present invention is described in more detail in the followingexamples.

EXAMPLE 1

Hyaluronic acid sodium salt (1×10 ⁻³ mol., with reference to thedisaccharidic unit) were transformed in TBA salt, according to one ofthe following methods:

a) 1% aqueous solution of sodium hyaluronate is transformed in H⁺form byH⁺cationic strong resin (Amberlite IR 120); the final solution istreated by a 0,5% solution of TBA-OH to about pH=9.

b) 1% aqueous solution of sodium hyaluronate is transformed in TBA saltsolution by treating with a cationic weak resin in TBA³⁰ form.(Amberlite IRC 50)

In both cases, the final solutions are liophylised. The TBA salt is thendissolved in 15 ml of anhydrous DMF, under N₂, and—at 0° C.—0, 02 g ofcloronethypyridylium Iodide (CMPJ) in 2 ml of anhydrous DFM, are addeddropwise to the stored solution of TBA.salt.

The reaction mixture was then added with 0.1 ml of triethylamine and,then, dropwise, with a solution of 1,3-diaminopropane (d=0.88, in largeexcess, so as to make cross-linking of the activated carboxy groupseasier) in 2 ml of anhydrous DMF. When the addition was over, thereaction mixture was stirred for at least 30′ and the solvent was thenremoved under reduced pressure, the residue was then taken up with DMF,which was subsequently removed by distillation; the residue was thentreated with ethanol, ethanol-water and finally with water.

The product was then lyophilised and the residue subjected to analysis.

I.R. (film): 1630 cm⁻¹ (—CO—NH); 1740 cm⁻¹ (—COOH, polysaccharide); 3200cm⁻¹ (—NH—).

SD (Swelling Degree, in water and r.t., after 15′; gravimetricdetermination; calculated according to:${{SD} = {\frac{W_{s} - {Wd}}{Wd} \cdot 100}},$

where:

W_(s)=weight of hydrated gel; Wd=weight of dry gel): 31.000

Cross-linking degree: 0.05 (5% of initially available carboxy groups).

EXAMPLE 2

According to the procedure and conditions reported in example 1, usingthe same HY and the same activating agent but 1,6-diaminohexane insteadof 1,3-diaminopropane, a cross-linked hyaluronic acid havingcross-linking degree of 0.05 was obtained.

I.R. (film): 1630 cm⁻¹ (—CO—NH); 1740 cm⁻¹ (—COOH polysaccharide); 3200cm⁻¹ (—NH—).

EXAMPLE 3

According to the procedure and conditions used in example 1, using as across-linking agent 0,0′-dis-(2-aminopropyl) PEG 500, a hyaluronic acidhaving a cross-linking degree of 0.05 was obtained.

I.R. (film): 1630 cm⁺¹ (—CO—NH); 1740 cm⁻¹ (—COOH polysaccharide); 3200cm⁻¹ (—NH—).

SD=31.000

EXAMPLE 4

0.6 g of hyaluronic acid tributylammonium salt (1×10³ mol., withreference to the disaccharide unit) were dissolved under stirring in 30ml of DMF under nitrogen. 0.08 g of chloromethylpyridylium iodide(3.5×10⁻⁴ mol) dissolved in 2 ml of DMF were added dropwise to thestirred solution kept at 0° C. The molar ratio was therefore about 3/1.

After 20 minutes 2 ml of 1,3-diaminopropane (0.024 mol) were added,followed immediately by 0.5 ml of triethylamine. A solid, gelatinousproduct was obtained, the product was then swelled with water and washedagain with ethanol.

The final product, after lyophilisation, shows at the scanningmicroscope an irregular pattern with smooth zones alternating to spongyzones.

The cross-linking degree was 0.3 (30% of initially available carboxygroups)

I.R. (film): 1740 cm⁻¹ (—COOH); 1630 cm⁻¹ (—CO—NH); 1610 cm⁻¹ (—COO—);1560 cm⁻¹ (—CO—NH—)

EXAMPLE 5

0.6 g of hyaluronic acid tributylammonium salt (HY TBA) (1×10⁻³ mol.,with reference to the disaccharide unit) were dissolved under stirringin 30 ml of DMF under nitrogen. 0.15 g of chloromethylpyridylium iodide(CMPJ) (6×10⁻⁶ mol) dissolved in 2 ml of DMF were added dropwise to thesolution, kept at 0° C. The molar ratio was 2HY.TBA:1 CMPJ. After 20minutes, 2 ml of 1,3 diaminopropane (0.024 mol.) were added to thesolution.

0.5 ml of triethylamine were added thereafter.

A solid, gelly-like product was obtained and thoroughly washed with DMF.

After evaporating DMF, the product was swelled in water and washed withethanol before lyophilization.

The obtained product had a cross-linking degree of 0.5 and showed at thescanning microscope a grainy aspect interspaced by large meshes. Athigher magnitudes, the two morphologies appear identical and showround-shaped protrusions a few microns in diameter.

IR (film): 1740 cm⁻¹ (—COOH); 1630 cm⁻¹ (—CO—NH—); 1610 cm⁻¹ (—COO);1560 cm⁻¹ (—CO—NH—);

The gels were subjected to swelling in PBS and the max swelling abilitywas evaluated.

SD=23.500

NMR=(13 C; ppm): 29.3 and 39.8

The rheological properties evaluated on Bohlin VOR Rheometer, at thetemperature of 23±0.1° C., show that the dynamic elastic module G′ (100Pa at 10 Hz) identical at the two considered concentrations (10 and 20mg/ml) is always higher than the viscous dynamic module (G″ 40 Pa for 20mg at 10 Hz and 20 Pa for 10 mg at 10 Hz).

EXAMPLES 6-9

According to the methods disclosed in the previous examples, thecross-linked hyaluronic acid derivatives having the characteristicssummarised in the following table 1, were obtained, starting from 1×10⁻³mol (0.6 g) of hyaluronic acid tributylammonium salt.

The obtained derivatives had the following properties

TABLE 1 Amount Cross- Aspect at Cross-linking (g) of linking I.R. thescanning Ex agent (mol) CMPJ (mol) degree SD NMR (13) (ppm) (film)(cm⁻¹) microscope 6 1,3-propane- diamine (0.024)  0.6 g (1.210⁻³) (100%)13.200

1630 (—CO—NH—); 1560 (—CO—NH—); Homogeneouns, ondulated morphology. 70,0′-1-bis-(2- 0.15 g  (50%) 9.000 Alternating smooth diaminopropyl) (6× 10⁻⁴) areas and meshes, PEG 500 circular protrusions (0.022) a fewmicrons in size. 8 0,0′-bis(2- 0.15 g  (50%) 6.100 Two morphologicallyaminopropyl) - (6 × 10⁻⁴) different zones, a PEG 800 first one ondulated(0.022) and a second with hole-like structures. 9 1,6-diamino- 0.15 g (50%) 8.000 169.46 1740 (—COOH); Smooth surface with hexane (6 × 10⁻⁴)(—CO—NH— of cross-linking); 1630 (—CO—NH—); protrusions having a (0.023)74.04/76.80/83.17/80.41 1610 (—COO⁻); few microns in size. (—CH2— ofcross-linking arm) 1560 (—CO—NH—);

EXAMPLE 10 Sulphation of 50% Cross-Linked HY

The derivative obtained in example 5 was dispersed in 5 ml DMF understrong stirring and nitrogen atmosphere.

A solution of 1 g of SO₃/pyridine in mol of DMF was added at 0° C. andstirred for 3 hours. The reaction was blocked by adding an excess of H₂O(50 ml) and the pH adjusted to 9 with 0.1M NaOH.

The product was thoroughly washed with ethanol and H₂O and thenlyophilized.

The IR spectrum shows, in addition to the bands of the starting product,a peak at 1260 cm⁻¹ and a stronger band at 1025 cm⁻¹.

The gel swells in PBS with SD=33.000. Higher resolution 13C NMR spectrumshows the signals in H₂O at 37° C. reported in table 2. The intensity ofthe NMR signals at 29.3 and 38.8 ppm (—CH₂—) and the signal at 172.5 ppm(CONH) confirm a cross-linking degree of about 50%.

The rheological properties are characterised by dynamic elastic modulesG′ (2500 Pa with 20 mg and 1000 Pa with 10 mg at 10 Hz) which are alwayshigher than the dynamic viscous modules G″ (600 Pa with 20 mg and 150 Pawith 10 mg at 10 Hz) and much higher than the corresponding valuesobtained with non-sulphated HY (13 at 50%—example 5). This compound hasa thrombin time (TT) higher (61±5″) than the control (14.0″) and thecorresponding not cross-linked (14.6″).

The compound was also active in the PRP test using stressed rabbit.

TABLE 2 Table: 13C Chemical shift C-1 C-2 C-3 C-4 C-5 x-C═O y-CH₃ 103.557.3 85.4 71.3 78.7 178.0 25.3 ppm C-1′ C-2′ C-3′ C-4′ C-5′ 6-C═O 105.975.2 76.4 82.8 78.6 176.2 ppm 1-CH2 2-CH2 3-CH2 6′-C═O CROSS- LINKING39.8 29.3 39.8 172.5 ppm

EXAMPLE 11

Using the same methodology, the sulphated derivatives of 50%cross-linked products according to example 7,8, and 9, have beensynthetized.

Colorimetric characteristics of the sulphated derivatives are reportedin table 3 together with that of the products deriving from examples 5and 10.

TABLE 3 CROSSLINKED POLYMER ΔHa Tg ΔHb Wt % (50% CROSS.LINKING DEGREE)[J/g] [° C.] [J/g] water C.L.Hyal-1,3 (Ex. 5) 276 51 42 12 C.L.HyalS-1,3(Ex. 10) 357 64 53 16 C.L.Hyal-1,6 (Ex. 9) 327 64 58 16 C.L.HyalS-1,6465 64 65 20 5 C.L.Hyal-P500.2NH₂ (Ex. 7) 239 45 72 10 6C.L.HyalS-P500.2NH₂ 384 69 113  16 7 C.L.Hyal-P800.2NH₂ (Ex. 8) 179 7330 10 8 C.L.HyalS-P800.2NH₂ 206 76 52 10 Hyal ITBA 164 — 130   5 ΔHa[J/g]: water vaporization henthalpy Tg [° C.]: enthalpy for thermaldegradation process ΔHb [J/g]: glass transition temperate Wt % water: %of water content, based on ΔHa

EXAMPLE 12 Preparation of Complexes of Cu, Zn and Fe

100 mg of lyophilized gel of the example 5 were added, under stirringand at room temperature, to 200 ml of a concentrated solution of copper(II) chloride in distilled water. The suspension was stirred for 24hours, and the complex was precipitated by addition of ethanol. Aftercentrifugation, the residue was washed repeatedly with water and ethanolto remove the excess ions.

The final gel, blue-green in color, was lyophilized and analyzed.

The same procedure was carried out using ZnCl₂ and FeCl₂.

The analysis (EDAX, polarography, HCl 0.1 N titration, atomicadsorption) shows a copper content of 0.5 mol/disaccharide units.

What is claimed is:
 1. A hyaluronic acid derivative comprisingcross-linked molecules of hyaluronic acid obtained by the reaction ofthe carboxylic acid groups of a hyaluronic acid wherein thecross-linkage occurs only through amide bonds between carboxy groups ofthe hyaluronic acid and the amino groups of a diamine of the formulaNH₂-A-NH₂ wherein A is a linear unsubstituted C₂-C₆ chain or apolyoxyalkylene chain of the formula [(CH₂—O—CH₂)₂]_(m) wherein m is aninterger from 2-10.
 2. A cross-linked hyaluronic acid derivativeaccording to claim 1 wherein the hydroxy groups of said hyaluronic acidderivative are sulphated or hemisuccinylated.
 3. A pharmaceuticalcomposition useful as (a) a substitute for synovial fluid in thetreatment, in which the principal active ingredient is a cross-linkedhyaluronic acid derivative according to claim 1.